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Article Specificity of the Parasitic flavipes (: Mymaridae) and a New Defence in Its Hosts (Coleoptera: Chrysomelidae: Oulema spp.)

Alena Samková 1,2,*, Jiˇrí Hadrava 2,3 , Jiˇrí Skuhrovec 4 and Petr Janšta 2 1 Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, CZ-165 00 Prague 6—Suchdol, Czech Republic 2 Department of Zoology, Faculty of Science, Charles University, Viniˇcná 7, CZ-128 43 Prague 2, Czech Republic; [email protected] (J.H.); [email protected] (P.J.) 3 Institute of , Biological Centre, Czech Academy of Sciences, Branišovská 31, CZ-370 05 Ceskˇ é Budˇejovice,Czech Republic 4 Group Function of Invertebrate and Plant Biodiversity in Agro-Ecosystems, Crop Research Institute, Drnovská 507, CZ-161 06 Praha 6—Ruzynˇe,Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-607-228-572

 Received: 17 January 2020; Accepted: 8 March 2020; Published: 10 March 2020 

Abstract: The parasitic wasp Anaphes flavipes (Förster, 1841) (Hymenoptera: Mymaridae) is an important of cereal leaf . Some species of cereal leaf co-occur in the same localities, but the host specificity of the wasp to these crop pests has not yet been examined in detail. A lack of knowledge of host specificity can have a negative effect on the use of this in biological control programs addressed to specific species or genus. In this study, laboratory experiments were conducted to assess the host specificity of A. flavipes for three species of cereal leaf beetles (Oulema duftschmidi Redtenbacher, 1874, Oulema gallaeciana Heyden, 1879 and Oulema melanopus Linnaeus, 1758) in central Europe. For the first time, a new host defence against egg occurring in O. gallaeciana from localities in the Czech Republic, a strong dark sticky layer on the egg surface, was found and described. The host specificity of A. flavipes was studied in the locality with the presence of this defence on O. gallaeciana (the dark sticky layer) (Czech Republic) and in a control locality (Germany), where no such host defence was observed. Contrary to the idea that a host defence mechanism can change the host specificity of parasitoids, the wasps from these two localities did not display any differences in that. Respectively, even though it has been observed that eggs with sticky dark layer can prevent parasitization, the overall rate of parasitization of the three species of cereal beetles has not been affected. However, in our view, new host defence can influence the effects of biological control, as eggs of all Oulema spp. in the locality are protected against parasitization from the wasps stuck on the sticky layer of the host eggs of O. gallaeciana.

Keywords: parasitoid-host interaction; biological control; host spectrum; Mymaridae

1. Introduction Parasitic wasps occur in high numbers, both in terms of species diversity and absolute numbers of individuals [1]. Despite differing estimates, the diversity of parasitic wasps is assumed to be over one million species, with roughly every tenth species of insects being a parasitic hymenopteran [1,2]. Parasitic wasps attack a wide range of hosts, and they play an important role in the biodiversity and balance of natural ecosystems and agriculture [3,4]. Especially in agriculture, parasitic wasps can be used to reduce important pest insects in biological control programmes [5]. Biological control can be implemented either as modification of landscaping strategies that support natural enemies

Insects 2020, 11, 175; doi:10.3390/insects11030175 www.mdpi.com/journal/insects Insects 2020, 11, 175 2 of 11

(conservation biological control) [6] or as releasing (once or repeatedly) of parasitoids or predators in the infested fields or areas (augmentative control) [7,8]. This latter strategy is effective for and natural protected areas, or if the pests acquire resistance to chemical spraying [9–12]. For example, for augmentative biological control, 170 species of parasitoids are used only in Europe [8]. In some cases, the effectiveness of biological control is up to 100%, e.g., the wasp Cosmocomoidea ashmeadi (Girault, 1915) (Hymenoptera: Mymaridae) controlling the leafhopper Homalodisca coagulata (Say, 1832) (: Cicadellidae) [13], or (Girault, 1928) (Hymenoptera: Mymaridae) against the weevil Gyllenhal, 1833 (Coleoptera: Curculionidae) [14,15]. Many parasitoids do not have 100% efficiency, and therefore, methods for improving the effectiveness of natural enemies or biological control have been sought [16]. The efficiency of biological control can be increased with detailed knowledge of the host specificity of parasitoids, and host specificity is one of the primary criteria for evaluating the risks of biological control organisms to nontarget organisms [17]. The host range is generally characterized as the set of species on which a control organism can feed and develop [17], all organisms in a given habitat are potentially a host for parasitoids, but their quality and parasitoid approbation is different [1]. The host may be attacked by one to over twenty parasitoids at a time, and the most vulnerable hosts to parasitization appear to be herbivorous insects [18]. Successful parasitization requires the parasitoid first to locate the host’s habitat, then perform a specific behavioral routine to finally lay eggs on or into the host [19]. Parasitoids are also capable of learning novel signals that improve their search efficiency [1]. The hosts are not merely passive participants in this process [3]. They have mechanical, physiological and immune defences against parasitoids and are in a constant evolutionary arms race; whatever defensive mechanisms the host invents, the parasitoid tries to overcome [20,21]. Our study is focused on the host specificity of the potential biological agent, Anaphes flavipes (Föster, 1841) (Hymenoptera: Mymaridae). The host spectrum of this wasp includes the rare spp. and the widespread Oulema spp. (O. duftschmidi Redtenbacher, 1874, O. gallaeciana Heyden, 1879, and O. melanopus Linnaeus, 1758) [22,23]. Larvae and adults of Oulema species damage the leaves of cereals (barley, wheat and oats), and they are an economically important crop pest in Europe and North America [24–26]. For example, in agricultural areas around the world, pests reduce grain crop yields by 5% to 20 % every year [27]. The use of parasitic wasps for biological control has been repeatedly tested [28–30]. In this context, the host spectrum of A. flavipes was examined for six taxons, Crioceris duodecimpunctata (Linnaeus, 1758); Oulema sayi (Crotch, 1873); Lema nigrovittata (Guérin-Méneville, 1844); Lema daturaphila Kogan and Goeden, 1970 (as L. trilineata (Olivier, 1808)); Lema trilineata californica (Schaefer Krauss 1947) and Lema trivittata trivittata Say, 1824) by Maltby et al. [31]; however, a current common host species, O. duftschmidi, was not included because, until 1989, it was assigned to O. melanopus [32]. In relation to the study of host specificity of A. flavipes, we describe a new type of host defense against egg parasitoid as a dark sticky layer on host eggs of O. gallaeciana in Czech localities. This sticky layer can completely prevent parasitization, because any females adhere to the sticky layer and are unable to either parasitize or release herself. The eggs defense of cereal beetles against parasitization by parasitoids has been proposed so far only by Anderson and Paschke [22] as a strong selective pressure on the rapid development of beetle larvae. The wasps reject host eggs older than 72 h, probably because the forming larvae could damage the parasitoid egg by sclerotized mandibles. However, there is no experimental evidence for this claim. First, laboratory experiments were carried out to assess the host specificity of Anaphes flavipes for three widespread Oulema species (O. duftschmidi, O. gallaeciana and O. melanopus). Secondly, we described a new type of host defence against parasitoids on eggs and compared the host specificity of the wasp A. flavipes between metapopulations with or without the observed egg defence on three co-occurring Oulema species. The main aim was to test whether the wasp’s choice of host species can be affected by the presence or absence of the host defence. Insects 2020, 11, 175 3 of 11

2. Materials and Methods

2.1. Parasitic Wasps Parasitic wasps (A. flavipes) were collected from host eggs of species O. duftschmidi, O. gallaeciana and O. melanopus in periods from the end of April until the end of June 2015–2016 in cereal fields in one locality in the Czech Republic (50.1385 N, 14.3695 E) and one locality in Germany (50.7787 N, 6.0381 E). The parasitized host eggs were stored in Petri dishes with moistened filter paper until the emergence of the adult wasps. These “wild” wasps were used as an initial population for rearing the next generations of parasitoids in an environmental chamber with conditions of 22 2 C, relative ± ◦ humidity of 40%–60% and 24 h light. All these “next generation” females and males of A. flavipes were bred in laboratory on the eggs Oulema species (O. duftschmidi and O. melanopus) and those used for experiments were at most 24 h old (post emergence). Each emerged female used in the experiment was immediately mated. Each mated female was then placed in a Petri dish with 12 host eggs (8 eggs of O. duftschmidi + O. melanopus, 4 eggs of O. gallaeciana). Before starting and during the experiments, the females were not fed, and they had constant access to water.

2.2. Host Species The host species of the genus Oulema (O. duftschmidi, O. gallaeciana and O. melanopus) were obtained from the adults collected in localities in the Czech Republic (one at the same location as that used for parasitic wasps and one more in Police nad Metují (50.5277 N, 16.2456 E)) and Germany (near the city of Aachen (50.7763 N, 6.0838 E)). The hosts were collected using a net or by hand collection. The Oulema species were divided into two groups: (1) O. gallaeciana (Og), and (2) O. duftschmidi (Od) + O. melanopus (Om). Although, O. gallaecina can be easily determined to species level using external morphological characters (such as body color), O. duftschmidi and O. melanopus are distinguishable only when the morphology of the genitals is applied, see [32]). Therefore each female of Od and Om which eggs have been used in experiments, were stored in ethanol and determined to species by genital preparation to enable assign host exact host species to each host egg used in the experiment. The Oulema species were bred in Petri dishes (diameter 8.5 cm, for pairs of hosts – Od + Om) or plastic boxes (10 10 5.5 cm or 20 20 18 cm, for more individuals – Og) with moistened filter paper in × × × × an environmental chamber at 22 2 C and a relative humidity of 40%–60%. The beetles were fed with ± ◦ grain leaves and had unlimited access to water. Cereal lay eggs on the fresh leaves of cereals. In our experiment, every 24 h, the leaves of cereals were removed from Petri dishes and plastic boxes, and fresh leaves were given. The leaves with eggs were cut into pieces which contain only one host egg (approximately 1 cm long piece of leave), each piece was numbered and placed into Petri dish (up a total of 12 eggs—4 Og and 8 Od + Om) for parasitation by one wasp.

2.3. Laboratory Experiments All laboratory experiments were performed in Petri dishes (diameter 8.5 cm) in a thermal cabinet at 22 2 C. Eggs were removed on the 9th or 10th day after parasitization, placed in 1.5 mL Eppendorf ± ◦ tubes and stored at the same temperature in the thermal cabinet. After wasps’ emergence, the number of parasitized host eggs by one female in relation to host species were measured.

2.3.1. Host Defence In 2012, at localities in the Czech Republic (50.5277 N, 16.2456 E; 50.1385 N, 14.3695 E), host eggs coated with a strong dark sticky layer (Figure1A,B) were observed for the first time. The parasitization of eggs with and without the dark sticky layer was documented by photography using a Canon EOS70D camera equipped with a Canon MP-E 65/2.8 MACRO lens. Insects 2020, 11, x 4 of 11

143 (nOg = 1083, nOd+Om = 3280) by one female of Oulema was measured in three categories: 1) sticky dark 144 (distinctive dark sticky layer; Figure 1A,B); 2) slightly sticky ( colour; the structure of the egg 145 surface is not visible through the sticky layer; Figure 1C,D); 3) non-sticky (yellow colour, the structure 146 of the egg surface is visible; Figure 1E,F) (Supplementary Material 1). The host eggs were Insects 2020, 11, 175 4 of 11 147 photographed with the Canon EOS70D camera (nOg = 500, nOd+Om = 500). The differences in the 148 proportion of eggs with the dark sticky layer between host species were tested by chi-square test.

149

150 FigureFigure 1. Three1. Three types types of Oulemaof Oulemaeggs: eggs: (A ,B(A), stickyB) sticky dark dark eggs eggs (distinctive (distinctive dark dark color; color; sticky sticky layer); layer); (C,D ) 151 (C,D) little sticky eggs (yellow color; the structure of the egg surface is not visible under the sticky little sticky eggs (yellow color; the structure of the egg surface is not visible under the sticky surface); 152 surface); (E,F) non-sticky eggs (yellow color; the structure of the egg surface is visible). Host eggs were (E,F) non-sticky eggs (yellow color; the structure of the egg surface is visible). Host eggs were not older 153 not older than 24 h. than 24 h. 154 2.3.2.In2016, Host OulemaSpecificityadults from these two localities (50.5277 N, 16.2456 E; 50.1385 N, 14.3695 E) were 155 collected,Each and female all individuals of A. flavipes were (n = 59) put from into the Petri two dishes localities (one with female Og eggs and with one malea sticky in layer one Petri (Czech dish 156 orRepublic; one female see in Supplementary one Petri dish) M andaterial divided 3 for details into two) had groups: 12 host (1) eggsO. (4 gallaeciana× Og with (aOg sticky)(nOg layer= 82) and and 157 (2)8O.× Od duftschmidi + Om without(Od) + aO. sticky melanopus layer) (availableOm)(nOd +forOm parasitization= 100). Every for 24 8 h h for in 15 a d,Petri the dish. number The ofhost eggs 158 laidspecificity (nOg = 1083, of thesenOd +waspsOm = 3280)was compared by one female to that of ofOulema the waspswas from measured control in German three categories: locality where (1) sticky no 159 darksticky (distinctive layer on Og dark eggs sticky was layer;observed Figure (see1 A,B);Supplementary (2) slightly material sticky (yellow4 for details colour;). Each the female structure of A. of 160 theflavipes egg surface (n = 18) is notfrom visible German through locality the had sticky 12 host layer; eggs Figure (4× 1OgC,D); and (3) 8× non-sticky Od + Om)(yellow available colour, for 161 theparasitization structure of for the 8 egg h in surface a Petri dish. is visible; The host Figure beetles1E,F) Om (Supplementary + Od (after laying Material host eggs) 1). were The stored host eggs in 162 96% ethanol and identified to species by their genitalia (see Host species). For each female, the total were photographed with the Canon EOS70D camera (nOg = 500, nOd+Om = 500). The differences in the 163 number of parasitized host eggs of the three host species (Od, Og, Om) was measured (Supplementary proportion of eggs with the dark sticky layer between host species were tested by chi-square test.

2.3.2. Host Specificity Each female of A. flavipes (n = 59) from the two localities with Og eggs with a sticky layer (Czech Republic; see Supplementary Material 3 for details) had 12 host eggs (4 Og with a sticky layer and 8 × × Od + Om without a sticky layer) available for parasitization for 8 h in a Petri dish. The host specificity of these wasps was compared to that of the wasps from control German locality where no sticky layer on Og eggs was observed (see Supplementary 4 for details). Each female of A. flavipes (n = 18) from Insects 2020, 11, x 5 of 11

164 Material 2). The wasps that stuck to the sticky surface of the eggs were discarded from the 165 experiment. 166 Insects 2020Control, 11, 175 locality in Germany: The Og females and males from Germany were collected5 ofat 11 the 167 same time as the parasitic wasps from the localities of cereals near the city of Aachen (50.7763N, 168 6.0838E). One female and one male or only one female were placed in Petri dishes with moistened German locality had 12 host eggs (4 Og and 8 Od + Om) available for parasitization for 8 h in a Petri 169 filter paper and crop leaves. Every× 24 h, the ×leaves of cereals with host eggs were removed from each 170 dish.Petri The dish host and beetles wereOm replaced+ Od (after by fresh laying leaves host eggs) and werewater. stored All obtained in 96% ethanol host eggs and identified(n = 45) were to 171 speciesphotographed by their genitalia using a Canon (see Host EOS70D species ).camera For each and female, were recorded the total category numberof ofparasitized sticky layer host (see eggsFigure 172 of the1). The three occurrence host species of (theOd ,darkOg, Om sticky) was layer measured on their (Supplementary eggs (Figure 1A,B) Material was 2).not The observed wasps. that stuck 173 to the stickyIn order surface to test of the the negative eggs were effect discarded of sticky from layer the on experiment. the host specificity of A. flavipes, for each A. 174 flavipesControl female, locality the in preference Germany: for The OdOg +females Om vs. andOg was males tested from by Germany a binomial were test. collected In this at case, the same a one- 175 timesided as the test parasitic was used wasps due to from the negative the localities effect of of cereals the dark near sticky the city layer of on Aachen the parasitization (50.7763 N,6.0838 of Og eggs. E). 176 OneFisher’s female method and one of male meta or analysis only one was female used werefor joining placed the in Petrip-values dishes (function with moistened “sumlog”filter in R paperpackage 177 and“metap” crop leaves. was used Every for 24 this h, the purpose) leaves. of Females cereals withfrom hostthe Czech eggs were Republic removed and fromGermany each Petriwere dishtested 178 andseparately were replaced due to by differences fresh leaves in and the water. presence All obtainedof the dark host sticky eggs (layern = 45) in were their photographed native ecosystems. using R 179 a Canonversion EOS70D 3.3.3 [33 camera] was used and werefor all recorded statistical category analyses. of sticky layer (see Figure1). The occurrence of the dark sticky layer on their eggs (Figure1A,B) was not observed. 180 3. ResultsIn order to test the negative effect of sticky layer on the host specificity of A. flavipes, for each A. flavipes female, the preference for Od + Om vs. Og was tested by a binomial test. In this case, a 181 one-sided3.1. Host test Defence was used due to the negative effect of the dark sticky layer on the parasitization of Og eggs. Fisher’s method of meta analysis was used for joining the p-values (function “sumlog” in R 182 The eggs of Og (n = 82) and Od + Om (n = 100) females were classified into the three categories package “metap” was used for this purpose). Females from the Czech Republic and Germany were 183 (1) sticky dark eggs (distinctive dark color; sticky layer); (2) little sticky eggs (yellow color; the tested separately due to differences in the presence of the dark sticky layer in their native ecosystems. 184 structure of the egg surface is not visible under the sticky surface) and (3) non-sticky eggs (yellow R version 3.3.3 [33] was used for all statistical analyses. 185 color; the structure of the egg surface is visible, Figure 1) for the locality in the Czech Republic. The 186 3. Resultseggs with a dark sticky layer (Figure 1A,B) were significantly more prevalent in the Og species (Figure 187 2A) than in Om and Od (Figure 2B) (Χ-squared test, neggsOg = 1083, neggsOd+Om = 3280, X-squared = 2857.5, 188 3.1.df Host = 2, Defencep < 0.001). 189 TheThe eggs behavior of Og ( nthat= 82) precedes and Od parasitization+ Om (n = 100) and females the parasitization were classified itself into is the documented three categories in Figures (1) 190 sticky3 and dark 4, which eggs (distinctive show the parasitization dark color; sticky of the layer); eggs (2) with little a stickysticky eggslayer. (yellow Three color;behavioral the structure situations 191 ofduring the egg the surface parasitization is not visible of Og under eggs thewith sticky a dark surface) sticky andlayer (3) (Figures non-sticky 1A,B) eggs from (yellow the Czech color; locality the 192 structurewere observed of the egg and surface described: is visible, Figure1) for the locality in the Czech Republic. The eggs with a 193 dark sticky(1) T layerhe female (Figure adheres1A,B) wereto the significantly sticky layer moreand is prevalent unable to in either the Og parasitizespecies (Figureor release2A) herself than in; 194 (2) The wasp is able to parasitize the egg but cannot release herself from the egg surface; Om and Od (Figure2B) (X-squared test, neggsOg = 1083, neggsOd+Om = 3280, X-squared = 2857.5, df = 2, 195 p < 0.001).(3) The wasp successfully parasitizes the eggs and leaves the host, but afterwards, she must clean 196 herself.

197 Figure 2. The graphs show the prevalence of three types of host eggs for O. gallaeciana (A) and O. 198 duftschmidiFigure 2+. TheO. melanopus graphs show(B) from the Czech prevalence Republic. of three types of host eggs for O. gallaeciana (A) and 199 O. duftschmidi + O. melanopus (B) from Czech Republic. The behavior that precedes parasitization and the parasitization itself is documented in Figures3 and 4, which show the parasitization of the eggs with a sticky layer. Three behavioral situations during Insects 2020, 11, 175 6 of 11

the parasitization of Og eggs with a dark sticky layer (Figure1A,B) from the Czech locality were observed and described:

(1) The female adheres to the sticky layer and is unable to either parasitize or release herself; (2) The wasp is able to parasitize the egg but cannot release herself from the egg surface; (3) The wasp successfully parasitizes the eggs and leaves the host, but afterwards, she must clean herself. Insects 2020, 11, x 6 of 11

200

201 FigureFigure 3. The 3. The parasitation parasitation on on the the non-sticky non-sticky eggseggs by A.A. flavipes flavipes: (:(A)A the) the female female lays lays own own eggs eggs into the into the 202 first eggfirst ofeggOulema of Oulemaspp. spp. (arrow (arrow indicates indicates the the );ovipositor); (B (B) )female female ends ends the the parasitization; parasitization; (C) female (C) female 203 examinesexamines thesuitability the suitability of the of secondthe second egg egg of Oulemaof Oulemaspp. spp. by by its its antennae; antennae; ( D(D)) the the female female layslays ownown eggs; 204 (E) femaleeggs; ( endsE) female the parasitizationends the parasitization (arrow ( indicatesarrow indicates the ovipositor). the ovipositor).

Insects 2020, 11, 175 7 of 11 Insects 2020, 11, x 7 of 11

205

206 Figure 4.4. The parasitation on the host eggs with dark sticky layer byby A.A. flavipesflavipes:(: (A–C) the dark sticky 207 layerlayer preventsprevents the the oviposition oviposition behavior behavior of A.of flavipesA. flavipes;(D;, E(D,E) the) waspsthe wasps are unable are unable to overcome to overcome the sticky the 208 layersticky of layer the hostof the eggs. host eggs.

209 3.2. Host Specificity 210 Change of the host specificity of A. flavipes wasps from localities with host defense presented as 211 a dark sticky layer on the host eggs of Og was not statistically confirmed (p = 0.99, p = 0.60, n = 59,

Insects 2020, 11, 175 8 of 11

3.2. Host Specificity Change of the host specificity of A. flavipes wasps from localities with host defense presented as Insects 2020, 11, x 8 of 11 a dark sticky layer on the host eggs of Og was not statistically confirmed (p = 0.99, p = 0.60, n = 59, respectively; meta-analysis on p-values from binomial tests, Figure5A). The German locality was used 212 respectively; meta-analysis on p-values from binomial tests, Figure 5A). The German locality was as a negative control due to the absence of Og eggs with a sticky layer (n = 18, Figure5B). 213 used as a negative control due to the absence of Og eggs with a sticky layer (n = 18, Figure 5B). 214

215 216 FigureFigure 5. 5.The The graphs graphs show show the the proportion proportion of of parasitized parasitized host host eggs eggs by byA. A. flavipes flavipeswasps wasps from from localities, localities, 217 wherewhere the the host host defense defense (host (host eggs eggs with with dark dark sticky sticky layer) layer) was was observed—Czech observed—Czech Republic Republic (A (),A and), and the the 218 localitieslocalities without without host host defense—Germany defense—Germany (B ().B). 4. Discussion 219 4. Discussion The parasitic wasp A. flavipes was introduced in the 1970s from Europe (France, Germany, Italy) to 220 The parasitic wasp A. flavipes was introduced in the 1970s from Europe (France, Germany, Italy) the USA [22,28], and the host spectrum of these wasps was examined for six species [31]. However, a 221 to the USA [22,28], and the host spectrum of these wasps was examined for six species [31]. However, common current host species, O. duftschmidi, was not included in the previous study because until 1989, 222 a common current host species, O. duftschmidi, was not included in the previous study because until it was assigned to O. melanopus [32]. However, O. duftschmidi occurs together with other crop beetles 223 1989, it was assigned to O. melanopus [32]. However, O. duftschmidi occurs together with other crop in the same grain field agroecosystems [34]. The host specificity of wasps for three common species 224 beetles in the same grain field agroecosystems [34]. The host specificity of wasps for three common of crop beetles (O. duftschmidi, O. gallaeciana or O. melanopus) was reviewed in central Europe. Our 225 species of crop beetles (O. duftschmidi, O. gallaeciana or O. melanopus) was reviewed in central Europe. experiments found that the A. flavipes populations from central Europe do not show host specificity due 226 Our experiments found that the A. flavipes populations from central Europe do not show host to the absence of any female preference for specific hosts of the genus Oulema. This confirmation may 227 specificity due to the absence of any female preference for specific hosts of the genus Oulema. This be partly due to the fact that the choice of a host normally depends on phylogeny and host [35], 228 confirmation may be partly due to the fact that the choice of a host normally depends on phylogeny which are both extremely similar among our three hosts. However, the finding that wasps parasitize 229 and host ecology [35], which are both extremely similar among our three hosts. However, the finding all three Oulema species without substantial preference is extremely beneficial for their practical use in 230 that wasps parasitize all three Oulema species without substantial preference is extremely beneficial biological control programmes. 231 for their practical use in biological control programmes. The effectiveness of biological control using parasitoids may be strongly affected by host defence. 232 The effectiveness of biological control using parasitoids may be strongly affected by host Many insect species are known to exhibit generally effective defence mechanisms to protect their eggs 233 defence. Many insect species are known to exhibit generally effective defence mechanisms to protect against parasitoids, e.g., thick egg chorions and oothecae or protective structures (e.g., scales, setae, 234 their eggs against parasitoids, e.g., thick egg chorions and oothecae or protective structures (e.g., feces, , spumaline; [3]). The rate of parasitization on an egg may be reduced by [3] 235 scales, setae, feces, silk, spumaline; [3]). The rate of parasitization on an egg may be reduced by or laying of the eggs in aggregation, where the protected eggs are in the middle [36] and below the 236 parental care [3] or laying of the eggs in aggregation, where the protected eggs are in the middle [36] surface layer [37]. Some species of the family Chrysomelidae deposit feces not only on their larvae but 237 and below the surface layer [37]. Some species of the family Chrysomelidae deposit feces not only on also on the surface of eggs as protection against parasitoids [38]. However, no host defence has been 238 their larvae but also on the surface of eggs as protection against parasitoids [38]. However, no host observed in eggs of Oulema species until now. 239 defence has been observed in eggs of Oulema species until now. Here, a new host defence mechanism of one Oulema species from Czech localities is demonstrated. 240 Here, a new host defence mechanism of one Oulema species from Czech localities is The eggs of all three studied Oulema species have a thin sticky layer that allows the eggs to stick to the 241 demonstrated. The eggs of all three studied Oulema species have a thin sticky layer that allows the leaves of grain and grass on which they are laid [39], but the eggs from some populations in the Czech 242 eggs to stick to the leaves of grain and grass on which they are laid [39], but the eggs from some Republic, which are significantly prevalent only in the species of O. gallaeciana, also have a strong sticky 243 populations in the Czech Republic, which are significantly prevalent only in the species of O. 244 gallaeciana, also have a strong sticky layer on their surface. A similar defence has also been observed 245 in with a sticky surface, which is a condition evolutionarily derived from galls without a sticky 246 layer [40]. In that case, the parasitoids attempting to lay their eggs into a developing insect in the 247 stick to the gall’s sticky surface [41]. Similarly, in the wasp A. flavipes, three situations that can happen 248 during the parasitization of the eggs with a dark sticky layer from the Czech locality were observed:

Insects 2020, 11, 175 9 of 11 layer on their surface. A similar defence has also been observed in galls with a sticky surface, which is a condition evolutionarily derived from galls without a sticky layer [40]. In that case, the parasitoids attempting to lay their eggs into a developing insect in the gall stick to the gall’s sticky surface [41]. Similarly, in the wasp A. flavipes, three situations that can happen during the parasitization of the eggs with a dark sticky layer from the Czech locality were observed:

(1) The female adheres to the sticky layer and is unable to either parasitize or release herself. First, before parasitization, the female needs to examine the suitability of the host eggs with her antennae (25; on average, for 12 s, n = 19 (Samková, unpubl.)). During this behavior, the wasp can adhere to the surface of the host egg before laying the eggs. In this case, the dark sticky layer succeeds in protecting the specific host egg. (2) The wasp is able to parasitize the egg but cannot unstick herself from the egg surface. Both of these host defence situations could be considered interspecific because other host eggs in the vicinity are protected against parasitization by this particular A. flavipes female. (3) The wasp successfully parasitizes and leaves the host, but afterwards, she must clean herself. At first, this defence may seem ineffective, because the sticky layer does not protect the egg from parasitization. However, this third observed behavior could lead to specialization in the wasps with such ‘experience’, which might afterwards prefer eggs without the dark sticky layer (such as those of O. duftschmidi and O. melanopus). It is known that, the choice of a host is related to the individual behavior and previous experiences of the female; flexible females could thus respond to a changing environment [19]. However, we must be careful in interpreting this claim, because scenario of wasps specialisation to host without defense againts parasitation is only our idea and future experiments are needed for it.

5. Conclusions In this study, we described a new host defence (dark sticky layer on the host eggs of O. gallaeciana) against egg A. flavipes. This was shown by the absence of significant proof of any differences in host preference between the wasps from the Czech localities, where the eggs of O. gallaeciana have a specific defence (dark sticky layer), and those from the German locality, where O. gallaeciana eggs have no known specific defence. However, the question remains whether biological control will be still as effective in localities with this host defence. We assume that the rate of can be reduced by the presence of eggs with the dark sticky layer, which often prevents the affected wasp from parasitizing other eggs. These claims, however, require the support of future field experiments.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4450/11/3/175/s1 Supplementary material 1 (1. Experiment - Host Defense for localities in Czech Republic); Supplementary material 2 (2. Experiment - Host specifity); Supplementary Material 3. (The eggs with dark sticky layer (Oulema gallaeciana Heyden, 1879 from Czech Republic)); Supplementary Material 4. (The eggs with little sticky and non-sticky layer (Oulema gallaeciana Heyden, 1879 from Germany)). Author Contributions: Data curation, A.S. and P.J.; formal analysis, J.H.; methodology, J.S., A.S. and P.J.; writing—original draft, A.S., J.H., J.S. and P.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Grant Agency of Charles University (GAUK) No. 243-227357 (to AS), by a grant of the Ministry of Education, Youth and Sports of the Czech Republic no. SVV 260434/2019 (to AS and PJ), Charles University Research Centre program No. 204069 (to PJ), and by the Ministry of Agriculture of the Czech Republic, institutional support MZe-RO0418 and from program NAZV No. QK1910281 (MZe CR)ˇ (to JS). Acknowledgments: We would like to thank the colleagues who lent us technical equipment: Petr Šípek, Tomáš Vendl, Zuzana Starostová, Dagmar Rˇ íhová, Petr Dolejš, and Pavel Just. We thank Jan Hrˇcek,Alois Honˇek and KateˇrinaVotýpková for valuable contributions to this work as well. We also thank Marek Romášek for language improvements. Conflicts of Interest: The authors declare no conflict of interest. Insects 2020, 11, 175 10 of 11

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